Augmentation and repair of vocal cord tissue defects

ABSTRACT

A method for corrective surgery in a human subject of a vocal cord defect amenable to rectification by the augmentation of tissue subadjacent to the vocal cord defect, the method comprising: placing an effective amount of autologous  in vitro  cultured cells into a vocal cord tissue of the subject in a position subadjacent to the vocal cord defect, wherein the vocal cord tissue is selected from the group consisting of a scar, Reinke&#39;s space, a muscle of the vocal cord, and the lamina propria, wherein the  in vitro  cultured cells are obtained by culturing a plurality of viable cells retrieved from the subject, and wherein the cultured cells are suspended in a collagen or modified collagen solution prior to injection of the cultured cells. The  in vitro  cultured cells can be fibroblasts, e.g., fibroblasts derived from dermis, fascia, connective tissue, or lamina propria.

This application is a division of co-pending U.S. Non-Provisionalapplication Ser. No. 09/003,378, filed Jan. 6, 1998, which claims thebenefit of U.S. Provisional Application Ser. No. 60/037,961 filed Feb.20, 1997.

FIELD OF INVENTION

The field of the present invention is the long-term augmentation and/orrepair of dermal, subcutaneous, or vocal cord tissue.

BACKGROUND OF INVENTION

I. In Vitro Cell Culture

The majority of in vitro vertebrate cell cultures are grown asmonolayers on an artificial substrate which is continuously bathed in anutrient medium. The nature of the substrate on which the monolayers maybe grown may be either a solid (e.g., plastic) or a semi-solid (e.g.,collagen or agar). Currently, disposable plastics have become apreferred substrate for cell culture.

While the growth of cells in two-dimensions is frequently used for thepreparation and examination of cultured cells in vitro, it lacks thecharacteristics of intact, in vivo tissue which, for example, includescell-cell and cell-matrix interactions. Therefore, in order tocharacterize these functional and morphological interactions, variousinvestigators have examined the use of three-dimensional substrates insuch forms as a collagen gel (Yang et al., Cancer Res. 41:1027 (1981);Douglas et al., In Vitro 16:306 (1980); Yang et al., Proc. Nat'l Acad.Sci. 2.088 (1980)), cellulose sponge (Leighton et al., J. Nat'l CancerInst. 12: 545 (1951)), collagen-coated cellulose sponge (Leighton etal., Cancer Res. 28: 286 (1968)), and GELFOAM® (Sorour et al., J.Neurosurg. 43:742 (1975)). Typically, these aforementionedthree-dimensional substrates are inoculated with the cells to becultured, which subsequently penetrate the substrate and establish a“tissue-like” histology similar to that found in vivo. Several attemptsto regenerate “tissue-like” histology from dispersed monolayers of cellsutilizing three-dimensional substrates have been reported. For example,three-dimensional collagen substrates have been utilized to culture avariety of cells including breast epithelium (Yang, Cancer Res. 41:1021(1981)), vascular epithelium (Folkman et al., Nature 288:551 (1980)),and hepatocytes (Sirica et al., Cancer Res. 76:3259 (1980)), howeverlong-term culture and proliferation of cells in such systems has not yetbeen achieved. Prior to the present invention, a three-dimensionalsubstrate had not been utilized in the autologous in vitro culture ofcells or tissues derived from the dermis, fascia, or lamina propria.

II. Augmentation and/or Repair of Dermal and Subcutaneous Tissues

In the practice of cosmetic and reconstructive plastic surgery it isfrequently necessary to employ the use of various injectable materialsto augment and/or repair defects of the subcutaneous or dermal tissue,thus effecting an aesthetic result. Non-biological injectable materials(e.g., paraffin) were first utilized to correct facial contour defectsas early as the late nineteenth century. However, numerous complicationsand the generally unsatisfactory nature of long-term aesthetic resultscaused the procedure to be rapidly abandoned. More recently, the use ofinjectable silicone became prevalent in the 1960's for the correction ofminor defects, although various inherent complications also limited theuse of this substance. Complications associated with the utilization ofinjectable liquid silicone include local and systemic inflammatoryreactions, formation of scar tissue around the silicone droplets,rampant and frequently-distant unpredictable migration throughout thebody, and localized tissue breakdown. Due to these potentialcomplications, silicone is not currently approved for general clinicaluse. Although the original proponents of silicone injection havecontinued experimental programs utilizing specially manufactured“Medical Grade” silicone (e.g., Dow Corning MDX 4.4011®) with a limitednumber of subjects, it appears highly unlikely that its use will begenerally adopted by the surgical community. See e.g., Spira and Rosen,Clin. Plastic Surgery 20:181 (1993); Matton et al., Aesthetic PlasticSurgery 9:133 (1985).

It has also been suggested to compound extremely small particulatespecies in a lubricious material and inject such combinationmicro-particulate media subcutaneously for both soft and hard tissueaugmentation and repair, however success has been heretofore limited.For example, bioreactive materials such as hydroxyapatite or cordalgranules (osteo conductive) have been utilized for the repair of hardtissue defects. Subsequent undesirable micro-particulate media migrationand serious granulomatous reactions frequently occur with the injectionof this material. These undesirable effects are well-documented with theuse of such materials as polytetrafluoroethylene (TEFLON®) spheres ofsmall diameter (˜90% of particles having a diameter of 30 μm) inglycerin. See e.g., Malizia et al., JAMA 251:3277 (1984). Additionally,the use of very small diameter particulate spheres (˜1-20 μm) or smallelongated fibrils (˜1-30 μm in diameter) of various materials in abiocompatible fluid lubricant as injectable implant composition aredisclosed in U.S. Pat. No. 4,803,075. However. while theseaforementioned materials create immediate augmentation and/or repair ofdefects, they also have a tendency to migrate and be reabsorbed from theoriginal injection site.

The poor results initially obtained with the use of non-biologicalinjectable materials prompted the use of various non-immunogenic,proteinaceous materials (e.g., bovine collagen and fibrin matrices).Prior to human injection, however, the carboxyl- and amino-terminalpeptides of bovine collagen must first be enzymatically-degraded, due toits highly immunogenic nature. Enzymatic degradation of bovine collagenyields a material (atelocollagen) which can be used in limitedquantities in patients pre-screened to exclude those who areimmunoreactive to this substance. The methodologies involved in thepreparation and clinical utilization of atelocollagen are disclosed inU.S. Pat. No. 3,949,073; U.S. Pat. No. 4,424,208; and U.S. Pat. No.4,488,911. Atelocollagen has been marketed as ZYDERM® brandatelocollagen solution in concentrations of 35 mg/ml and 65 mg/ml.Although atelocollagen has been widely employed, the use of ZYDERM® hasbeen associated with the development of anti-bovine antibodies inapproximately 90% of patients and with overt immunologic complicationsin 1-3% of patients. See DeLustro et al., Plastic and ReconstructiveSurgery 79:581 (1987).

Injectable atelocollagen solution also was shown to be absorbed from theinjection site, without replacement by host material, within a period ofweeks' to months. Clinical protocols calling for repeated injections ofatelocollagen are, in practice, primarily limited by the development ofimmunogenic reactions to the bovine collagen. In order to mitigate theselimitations, bovine atelocollagen was further processed by cross-linkingwith 0.25% glutaraldehyde, followed by filtration and mechanicalshearing through fine mesh. The methodologies involved in thepreparation and clinical utilization of this material are disclosed inU.S. Pat. No. 4,582,640 and U.S. Pat. No. 4,642,117. The modifiedatelocollagen was marketed as ZYPLAST® brand cross-linked bovineatelocollagen. The propertied advantages of cross-linking was to provideincreased resistance to host degradation, however this was off-set by anincrease in solution viscosity. In addition, cross-linking of the bovineatelocollagen was found to decrease the number of host cells whichinfiltrated the injected collagen site. The increased viscosity, and inparticular irregular increased viscosity resulting in “lumpiness,” notonly rendered the material, more difficult to utilize, but also made itunsuitable for use in certain circumstances. See e.g., U.S. Pat. No.5,366,498. In addition, several investigators have reported that thereis no or marginally-increased resistance to host degradation of ZYPLAST®cross-linked bovine atelocollagen in comparison to that of thenon-cross-linked ZYDERM® atelocollagen and that the overall longevity ofthe injected material is, at best, only 4-6 months. See e.g., Ozgentaset al., Ann. Plastic Surgery 33:171 (1994); and Matti and Nicolle,Aesthetic Plastic Surgery 14:227 (1990). Moreover, bovine atelocollagencross-linked with glutaraldehyde may retain this agent as a highmolecular weight polymer which is continuously hydrolyzed, thusfacilitating the release of monomeric glutaraldehyde. The monomeric formof glutaraldehyde is detectable in body tissues for up to 6 weeks afterthe initial injection of the cross-linked atelocollagen. The cytotoxiceffect of glutaraldehyde on in vitro fibroblast cultures is indicativeof this substance not being an ideal cross-linking agent for a dermalequivalent which is eventually infiltrated by host cells and in whichthe bovine atelocollagen matrix is rapidly degraded, thus resulting inthe release of monomeric glutaraldehyde 5 into the bodily tissues andfluids.

Similarly, chondroitin-6-sulfate (GAG), which weakly binds to collagenat neutral pH, has also been utilized to chemically modify bovineprotein for tissue graft implantation. See Hansborough and Boyce, JAMA136:2125 (1989). However, like glutaraldehyde, GAG may be released intothe tissue causing unforeseen long-term effects on human subjects. GAGhas been reported to increase scar tissue formation in wounds, which isto be avoided in grafts. Additionally, a reduction of collagen bloodclotting capacity may also be deleterious in the application in bleedingwounds, as fibrin clot contributes to an adhesion of the graft to thesurrounding tissue.

The limitations which are imposed by the immunogenicity of both modifiedand non-modified bovine atelocollagen have resulted in the isolation ofhuman collagen from placenta (see e.g., U.S. Pat. No. 5,002,071); fromsurgical specimens (see e.g., U.S. Pat. No. 4,969,912 and U.S. Pat. No.5,332,802); and cadaver (see e.g., U.S. Pat. No. 4,882,166). Moreover,processing of human-derived collagen by cross-linking and similarchemical modifications is also required, as human collagen is subject toanalogous degradative processes as is bovine collagen. Human collagenfor injection, derived from a sample of the patient's own tissue, iscurrently available and is marketed as AUTOLOGEN®. It should be noted,however, that there is no quantitative evidence which demonstrates thathuman collagen injection results in lower levels of implant degradationthan that which is found with bovine collagen preparations. Furthermore,the utilization of autologous collagen preparation and injection islimited to those individuals who have previously undergone surgery, dueto the fact that the initial culture from which the collagen is producedis derived is from the tissue removed during the surgical procedure.Therefore, it is evident that, although human collagen circumvents thepotential for immunogenicity exhibited by bovine collagen, it fails toprovide long-term therapeutic benefits and is limited to those patientwho have undergone prior surgical procedures.

An additional injectable material currently in use as an alternative toatelocollagen augmentation of the subjacent dermis consists of a mixtureof gelatin powder, -aminocapronic acid, and the patient's plasmamarketed as FIBREL®. See Multicenter Clinical Trial, J. Am. Acad.Dermatology 16:1155 (1987). The action of FIBREL® appears to bedependent upon the initial induction of a sclerogenic inflammatoryresponse to the augmentation of the soft tissue via the subcutaneousinjection of the material. See e.g., Gold, J. Dermatologic Surg.Oncology, 20:586 (1994). Clinical utilization of FIBREL® has beenreported to often result in an overall lack of implant uniformity (i.e.,“lumpiness”) and longevity, as well as complaints of patient discomfortassociated with its injection. See e.g., Millikan et al., J.Dermatologic. Surg. Oncology, 17:223 (1991). Therefore, in conclusion,none of the currently utilized protein-based injectable materialsappears to be totally satisfactory for the augmentation and/or repair ofthe subjacent dermis and soft tissue.

The various complications associated with the utilization of theaforementioned materials have prompted experimentation with theimplantation (grafting) of viable, living tissue to facilitateaugmentation and/or repair of the subjacent dermis and soft tissue. Forexample, surgical correction of various defects has been accomplished byinitial removal and subsequent re-implantation of the excised adiposetissue either by injection (see e.g., Davies et al., Arch. ofOtolarynaology-Head and Neck Surgery 121:95 (1995); McKinney & Pandya,Aesthetic Plastic Surgery 18: 383 (1994); and Lewis, Aesthetic PlasticSurgery 17:109 (1993)) or by the larger scale surgical-implantation (seee.g., Ersck, Plastic & Reconstructive Surgery 87: 219 (1991)). Toperform both of the aforementioned techniques a volume of adipose tissueequal or greater than is required for the subsequent augmentation orrepair procedure must be removed from the patient. Thus, for large scalerepair procedures (e.g., breast reconstruction) the amount of adiposetissue which can be surgically-excised from the patient may be limiting.In addition, other frequently encountered difficulties with theaforementioned methodologies include non-uniformity of the injectate,unpredictable longevity of the aesthetic effects, and a 4-6 week periodof post-injection inflammation and swelling. In contrast, in a preferredembodiment, the present invention utilizes the surgical engraftment ofautologous adipocytes which have been cultured on a solid supporttypically derived from, but not limited to, collagen or isolatedextracellular matrix. The culture may be established from a simple skinbiopsy specimen and the amount of adipose tissue which can besubsequently cultured in vitro is not limited by the amount of adiposetissue initially excised from the patient.

Living skin equivalents have been examined as a methodology for therepair and/or replacement of human skin. Split thickness autographs,epidermal autographs (cultured autogenic keratinocytes), and epidermalallographs (cultured allogenic keratinocytes) have been used with avarying degree of success. However, unfortunately, these forms oftreatment have all exhibited numerous disadvantages. For example, splitthickness autographs generally show limited tissue expansion, requirerepeated surgical operations, and give rise to unfavorable aestheticresults. Epidermal autographs require long periods of time to becultured, have a low success (“take”) rate of approximately 30-48%,frequently form spontaneous blisters, exhibit contraction to 60-70% oftheir original size, are vulnerable during the first 15 days ofengraftment, and are of no use in situations where there is bothepidermal and dermal tissue involvement. Similarly, epidermal allografts(cultured allogenic keratinocytes) exhibit many of the limitations whichare inherent in the use of epidermal autographs. Additionalmethodologies have been examined which involve the utilization ofirradiated cadaver dermis. However, this too has met with limitedsuccess due to, for example, graft rejection and unfavorable aestheticresults.

Living skin equivalents comprising a dermal layer of rodent fibroblastcells cast in soluble collagen and an epidermal layer of cultured rodentkeratinocytes have been successfully grafted as allografts onto SpragueDawley rats by Bell et al., J. Investigative Dermatology 81:2 (1983).Histological examination of the engrafted tissue revealed that theepidermal layer had fully differentiated to form desmonosomes,tonofilaments, keratohyalin, and a basement lamella. However, subsequentattempts to reproduce the living skin equivalent using human fibroblastsand keratinocytes has met with only limited success. In general, thekeratinocytes failed to fully differentiate to form a basement lamellaand the dermo-epidermal junction was a straight line.

The present invention includes the following methodologies for therepair and/or augmentation of various skin defects: (1) the injection ofautologously cultured dermal or fascial fibroblasts into various layersof the skin or injection directly into a “pocket” created in the regionto be repaired or augmented, or (2) the surgical engraftment of“strands” derived from autologous dermal and fascial fibroblasts whichare cultured in such a manner as to form a three-dimensional“tissue-like” structure similar to that which is found in vivo.Moreover, the present invention also differs on a two-dimensional levelin that “true” autologous culture and preparation of the cells isperformed by utilization of the patient's own cells and serum for invitro culture.

III. Vocal Cord Tissue Augmentation and/or Repair

Phonation is accomplished in humans by the passage of air past a pair ofvocal cords located within the larynx. Striated muscle fibers within thelarynx, comprising the constrictor muscles, function so as to vary thedegree of tension in the vocal cords, thus regulating both their overallrigidity and proximity to one another to produce speech. However, whenone (or both) of the vocal cords becomes totally or partially immobile,there is a diminution in the voice quality due to an inability toregulate and maintain the requisite tension and proximity of the damagedcord in relation to that of the operable cord. Vocal cord paralysis maybe caused by cancer, surgical or mechanical trauma, or similarafflictions which render the vocal cord incapable of being properlytensioned by the constrictor muscles.

One therapeutic approach which has been examined to allow phonationinvolves the implantation or injection of biocompatible materials. Ithas long been recognized that a paralyzed or damaged vocal cord may berepositioned or supported so as to remain in a fixed location relativeto the operable cord such that the unilateral vibration of the operablecord produces an acceptable voice pattern. Hence, various surgicalmethodologies have been developed which involve the formation of anopening in the thyroid cartilage and subsequently providing a means forthe support and/or repositioning of the paralyzed vocal cord.

For example, injection of TEFLON® into the paralyzed vocal cord toincrease its inherent “bulk” has been described. See e.g., von Leden etal., Phonosurgery 3:175 (1989). However, this procedure is nowconsidered unacceptable due to the inability of the injected TEFLON® toclose large glottic gaps, as well as its tendency to induce inflammatoryreactions resulting in the formation of fibrous infiltration into theinjected cord. See e.g., Maves et al., Phonosurgery: Indications andPitfalls 98:577 (1989). Moreover, removal of the injected TEFLON® may bequite difficult should it subsequently be desired or become necessary.

Another methodology for supporting the paralyzed vocal cord which hasbeen employed involves the utilization of a custom-fitted block ofsiliconized rubber (SILASTIC®). In order to ensure the proper fit of theimplant, the surgeon hand carves the SILASTIC® block during theprocedure in order to maximize the ability of the patient to phonate Thepatient is kept under local anesthesia so that he or she can producesounds to test the positioning of the implant. Generally, the implantedblocks are formed into the shape of a wedge which is totally implantedwithin the thyroid cartilage or a flanged plug which can be movedback-and-forth within the opening in the thyroid cartilage to fine-tunethe voice of the patient.

Although SILASTIC® implants have proved to be superior over TEFLON®injections, there are several areas of dissatisfaction with theprocedure including difficulty in the carving and insertion of theblock, the large amount of time required for the procedure, and a lackof an efficient methodology for locking the block in place within thethyroid cartilage. In addition, vocal cord edema, due to the prolongednature of the procedure and repeated voice testing during the operation,may also prove problematic in obtaining optimal voice quality.

Other methodologies which have been utilized in the treatment of vocalcord paralysis and damage include GELFOAM® hydroxyapatite, and porousceramic implants, as well as injections of silicone and collagen. Seee.g., Koufman, Larynagolastic Phonosurgery (1988). However, thesematerials have also proved to be less than ideal due to difficulties inthe sizing and shaping of the solid implants as well as the potentialfor subsequent immunogenic reactions. Therefore, there still remains aneed for the development of a methodology which allows the efficacioustreatment of vocal cord paralysis and/or damage.

SUMMARY OF THE INVENTION

The present invention discloses a methodology for the long-termaugmentation and/or repair of dermal, subcutaneous, or vocal cord tissueby the injection or direct surgical placement/implantation of: (1)autologous cultured fibroblasts derived from connective tissue, dermis,or fascia; (2) lamina propria tissue; (3) fibroblasts derived from thelamina propria; or (4) adipocytes. The fibroblast cultures utilized forthe augmentation and/or repair of skin defects are derived from eitherconnective tissue, dermal, and/or fascial fibroblasts. Typical defectsof the skin which can be corrected with the injection or direct surgicalplacement of autologous fibroblasts or adipocytes include rhytids,stretch marks, depressed scars, cutaneous depressions of traumatic ornon-traumatic origin, hypoplasia of the lip, and/or scarring from acnevulgaris. Typical defects of the vocal cord which can be corrected bythe injection or direct surgical placement of lamina propria orautologous cultured fibroblasts from lamina propria include scarred,paralyzed, surgically or traumatically injured, or congenitallyunderdeveloped vocal cord(s).

The use of autologous cultured fibroblasts derived from the dermis,fascia, connective tissue, or lamina propria mitigates the possibilityof an immunogenic reaction due to a lack of tissue histocompatibility.This provides vastly superior post-surgical results. In a preferredembodiment of the present invention, fibroblasts of connective tissue,dermal, or facial origin as well as adipocytes are derived fromfull-thickness biopsies of the skin. Similarly, lamina propria tissue orfibroblasts derived from the lamina propria are obtained from vocal cordbiopsies. It should be noted that the aforementioned tissues are derivedfrom the individual who will subsequently undergo the surgicalprocedure, thus mitigating the potential for an immunogenic reaction.These tissues are then expanded in vitro utilizing standard tissueculture methodologies.

Additionally, the present invention further provides a methodology ofrendering the cultured cells substantially free of potentiallyimmunogenic serum-derived proteins by late-stage passage of the culturedfibroblasts, lamina propria tissue, or adipocytes in serum-free mediumor in the patient's own serum. In addition, immunogenic proteins may bemarkedly reduced or eliminated by repeated washing in phosphate-bufferedsaline (PBS) or similar physiologically-compatible buffers.

DESCRIPTION OF THE INVENTION

I. Histology of the Skin

The skin is composed of two distinct layers: the epidermis, aspecialized epithelium derived from the ectoderm, and beneath this, thedermis, of vascular dense connective tissue, a derivative of mesoderm.These two layers are firmly adherent to one another and form a regionwhich varies in overall thickness from approximately 0.5 to 4 mm indifferent areas of the body. Beneath the dermis is a layer of looseconnective tissue which varies from areolar to adipose in character.This is the superficial fascia of gross anatomy, and is sometimesreferred to as the hypodermis, but is not considered to be part of theskin. The dermis is connected to the hypodermis by connective tissuefibers which pass from one layer to the other.

A. Epidermis

The epidermis, a stratified squamous epithelium, is composed of cells oftwo separate and distinct origins. The majority of the epithelium, ofectodermal origin, undergoes a process of keratinization resulting inthe formation of the dead superficial layers of skin. The secondcomponent comprises the melanocytes which are involved in the synthesisof pigmentation via melanin. The latter cells do not undergo the processof keratinization. The superficial keratanized cells are continuouslylost from the surface and must be replaced by cells that arise from themitotic activity of cells of the basal layers of the epidermis. Cellswhich result from this proliferation are displaced to higher levels, andas they move upward they elaborate keratin, which eventually replacesthe majority of the cytoplasm. As the process of keratinizationcontinues the cell dies and is finally shed. Therefore, it should beappreciated that the structural organization of the epidermis intolayers reflects various stages in the dynamic process of cellularproliferation and differentiation.

B. Dermis

It is frequently difficult to quantitatively differentiate the limits ofthe dermis as it merges into the underlying subcutaneous layer(hypodermis). The average thickness of the dermis varies from 0.5 to 3mm and is further subdivided into two strata—the papillary layersuperficially and the reticular layer beneath. The papillary layer iscomposed of thin collagenous, reticular, and elastic fibers arranged inan extensive network. Just beneath the epidermis, reticular fibers ofthe dermis form a close network into which the basal processes of thecells of the stratum germinativum are anchored. This region is referredto as the basal lamina.

The reticular layer is the main fibrous bed of the dermis. Generally,the papillary layer contains more cells and smaller and finer connectivetissue fibers than the reticular layer. It consists of coarse, dense,and interlacing collagenous fibers, in which are intermingled a smallnumber of reticular fibers and a large number of elastic fibers. Thepredominant arrangement of these fibers is parallel to the surface ofthe skin. The predominant cellular constituent of the dermis arefibroblasts and macrophages. In addition, adipose cells may be presenteither singly or, more frequently, in clusters. Owing to the directionof the fibers, lines of skin tension, Langer's lines, are formed. Theoverall direction of these lines is of surgical importance sinceincisions made parallel with the lines tend to gape less and heal withless scar tissue formation than incisions made at right-angles orobliquely across the lines. Pigmented, branched connective tissue cells,chromatophores, may also be present. These cells do not elaboratepigment but; instead, apparently obtain it from melanocytes.

Smooth muscle fibers may also be found in the dermis. These fibers arearranged in small bundles in connection with hair follicles (arrectorespilorum muscles) and are scattered throughout the dermis in considerablenumbers in the skin of the nipple, penis, scrotum, and parts of theperineum. Contraction of the muscle fibers gives the skin of theseregions a wrinkled appearance. In the face and neck, fibers of someskeletal muscles terminate in delicate elastic fiber networks of thedermis.

C. Adipose Tissue/Adipocytes

Fat cells, or adipocytes, are scattered in areolar connective tissue.When adipocytes form large aggregates, and are the principle cell type,the tissue is designated adipose tissue. Adipbcytes are fullydifferentiated cells and are thus incapable of undergoing mitoticdivision. New adipocytes therefore, which may develop at any time withinthe connective tissue, arise as a result of differentiation of moreprimitive cells. Although adipocytes, prior to the storage of lipid,resemble fibroblasts, it is likely that they arise directly fromundifferentiated mesenchymal tissue.

Each adipocyte is surrounded by a web of fine reticular fibers; in thespaces between are found fibroblasts, lymphoid cells, eosinophils, andsome mast cells. The closely spaced adipocytes form lobules, separatedby fibrous septa. In addition, there is a rich network of capillaries inand between the lobules. The richness of the blood supply is indicativeof the high rate of metabolic activity of adipose tissue.

It should be appreciated that adipose tissue is not static. There is adynamic balance between lipid deposit and withdrawal. The lipidcontained within adipocytes may be derived from three sources.Adipocytes, under the influence of the hormone insulin, can synthesizefat from carbohydrate. They can also produce fat from various fattyacids which are derived from the initial breakdown of dietary fat. Fattyacids may also be synthesized from glucose in the liver and transportedto adipocytes as serum lipoproteins. Fats derived from different sourcesalso differ chemically. Dietary fats may be saturated or unsaturated,depending upon the individual diet. Fat which is synthesized fromcarbohydrate is generally saturated. Withdrawals of fat result fromenzymatic hydrolysis of stored fat to release fatty acids into the bloodstream. However, if there is a continuous supply of exogenous glucose,then fat hydrolysis is negligible. The normal homeostatic balance isaffected by hormones, principally insulin, and by the autonomic nervoussystem, which is responsible for the mobilization of fat from adiposetissue.

Adipose tissue may develop almost anywhere areolar tissue is prevalent,but in humans the most common sites of adipose tissue accumulation arethe subcutaneous tissues (where it is referred to as the panniculusadiposus), in the mesenteries and omenta, in the bone marrow, andsurrounding the kidneys. In addition to its primary function of storageand metabolism of neutral fat, in the subcutaneous tissue, adiposetissue also acts as a shock absorber and insulator to prevent excessiveheat loss or gain through the skin.

II. Histology of the Larynx and Vocal Cords

The larynx is that part of the respiratory system which connects thepharynx and trachea. In addition to its function as part of therespiratory system, it plays an important role in phonation (speech).The wall of the larynx is composed of a “skeleton” of hyaline andelastic cartilages, collagenous connective tissue, striated muscle, andmucous glands. The major cartilages of the larynx (the thyroid, cricoid,and arytenoids) are hyaline, whereas the smaller cartilages (thecorniculates, cuneiforms, and the tips of the arytenoids) are elastic,as is the cartilage of the epiglottis. The aforementioned cartilages,together with the hyoid bone, are connected by three large, flatmembranes: the thyrohyoid, the quadrates, and the cricovocal. These arecomposed of dense fibroconnective tissue in which many elastic fibersare present, particularly in the cricovocal membrane. The true and falsevocal cords (vocal and vestibular ligaments) are, respectively, the freeupper boarders of the cricovocal (cricothyroid) and the free lowerboarders of the quadrate (aryepiglottic) membranes. Extending laterallyon each side between the true and false cords are the sinus and sacculeof the larynx, a small slit-like diverticulum. Behind the cricoid andarytenoid cartilages, the posterior wall of the pharynx is formed by thestriated muscle of the pharyngeal constrictor muscles.

The epithelium of the mucous membrane of the larynx varies withlocation. For example, over the vocal folds, the lamina propria of thestratified squamous epithelium is extremely dense and firmly bound tothe underlying connective tissue of the vocal ligament. While there isno true submucosa in the larynx, the lamina propria of the mucousmembrane is thick and contains large numbers of elastic fibers.

III. Methodologies

A. In Vitro Cell Culture of Fibroblasts or Lamina Propria

While the present invention may be practiced by utilizing any type ofnon-differentiated mesenchymal cell found in the skin which can beexpanded in in vitro culture, fibroblasts derived from dermal,connective tissue, fascial, lamina proprial tissues, adipocytes, and/orextracellular tissues derived from the cells are utilized in a preferredembodiment due to their relative ease of isolation and in vitroexpansion in tissue culture. In general, tissue culture techniques whichare suitable for the propagation of non-differentiated mesenchymal cellsmay be used to expand the aforementioned cells/tissue and practice thepresent invention as further discussed below. See e.g., Culture ofAnimal Cells: A Manual of Basic Techniques, Freshney, R. I. ed., (AlanR. Liss & Co., New York 1987); Animal Cell Culture: A PracticalApproach, Freshney, R. I. ed., (IRL Press, Oxford, England 1986), whosereferences are incorporated herein by reference.

The utilization of autologous engraftment is a preferred therapeuticmethodology due to the potential for graft rejection associated with theuse of allograft-based engraftment. Autologous grafts (i.e., thosederived directly from the patient) ensure histocompatibility byinitially obtaining a tissue sample via biopsy directly from the patientwho will be undergoing the corrective surgical procedure and thensubsequently culturing fibroblasts derived from the dermal, connectivetissue, fascial, or lamina proprial regions contained therein.

While the following sections will primarily discuss the autologousculture of fibroblasts of connective tissue, dermal, or fascial origins,in vitro culture of lamina propria tissue may also be establishedutilizing analogous methodologies. An autologous fibroblast culture ispreferably initiated by the following methodology. A full-thicknessbiopsy of the skin (−3×6 mm) is initially obtained through, for example,a punch biopsy procedure. The specimen is repeatedly washed withantibiotic and anti-fungal agents prior to culture. Through a process ofsterile microscopic dissection, the keratinized tissue-containingepidermis and subcutaneous adipocyte-containing tissue is removed, thusensuring that the resultant culture is substantially free ofnon-fibroblast cells (e.g., adipocytes and keratinocytes). The isolatedadipocytes-containing tissue may then be utilized to establish adipocytecultures. Alternately, whole tissue may be cultured andfibroblast-specific growth medium may be utilized to “select” for thesecells.

Two methodologies are generally utilized for the autologous culture offibroblasts in the practice of the present invention—mechanical andenzymatic. In the mechanical methodology, the fascia, dermis, orconnective tissue is initially dissected out and finely divided withscalpel or scissors. The finely minced pieces of the tissue areinitially placed in 1-2 ml of medium in either a 5 mm petri dish(Costar), a 24 multi-well culture plate (Corning), or other appropriatetissue culture vessel. Incubation is preferably performed at 37° C. in a5% CO₂ atmosphere and the cells are incubated until a confluentmonolayer of fibroblasts has been obtained. This may require up to 3weeks of incubation. Following the establishment of confluence, themonolayer is trypsinized to release the adherent fibroblasts from thewalls of the culture vessel. The suspended cells are collected bycentrifugation, washed in phosphate-buffered saline, and resuspended inculture medium and placed into larger culture vessels containing theappropriate complete growth medium.

In a preferred embodiment of the enzymatic culture methodology, piecesof the finely minced tissue are digested with a protease for varyingperiods of time. The enzymatic concentration and incubation time arevariable depending upon the individual tissue source, and the initialisolation of the fibroblasts from the tissue as well as the degree ofsubsequent outgrowth of the cultured cells are highly dependent uponthese two factors. Effective proteases include, but are not limited to,trypsin, chymotrypsin, papain, chymopapain, and similar proteolyticenzymes. Preferably, the tissue is incubated with 200-1000 U/ml ofcollagenase type II for a time period ranging from 30-minutes to 24hours, as collagenase type II was found to be highly efficacious inproviding a high yield of viable fibroblasts. Following enzymaticdigestion, the cells are collected by centrifugation and resuspendedinto fresh medium in culture flasks.

Various media may be used for the initial establishment of an in vitroculture of human fibroblasts. Dulbecco's Modified Eagle Medium (DMEM,Gibco/BRL Laboratories) with concentrations of fetal bovine serum (FBS),cosmic calf serum (CCS), or the patient's own serum varying from 5-20%(v/v)—with higher concentrations resulting in faster culture growth—arereadily utilized for fibroblast culture. It should be noted thatsubstantial reductions in the concentration of serum (i.e., 0.5% v/v)results in a loss of cell viability in culture. In addition, thecomplete culture medium typically contains L-glutamine, sodiumbicarbonate, pyridoxine hydrochloride, 1 g/liter glucose, and gentamycinsulfate. The use of the patient's own-serum mitigates the possibility ofsubsequent immunogenic reaction due to the presence of constituentantigenic proteins in the other serums.

Establishment of a fibroblast cell line from an initial human biopsyspecimen generally requires 2 to 3.5 weeks in total. Once the initialculture has reached confluence, the cells may be passaged into newculture flasks following trypsinization by standard methodologies knownwithin the relevant field. Preferably, for expansion, cultures are“split” 1:3 or 1:4 into T-150 culture flasks (Corning) yielding ˜5×10⁷cells/culture vessel. The capacity of the T-150 culture flask istypically reached following 5-8 days of culture at which time thecultured cells are found to be confluent.

Cells are preferably removed for freezing and long-term storage duringthe early passage stages of culture, rather than the later stages due tothe fact that human fibroblasts are capable of undergoing a finitenumbers of passages. Culture medium containing 70% DMEM growth medium,10% (v/v) serum, and 20% (v/v) tissue culture grade dimethylsulfoxide(DMSO, Gibco/BRL) may be effectively utilized for freezing of fibroblastcultures. Frozen cells can subsequently be used to inoculate secondarycultures to obtain additional fibroblasts for use in the originalpatient, thus doing away with the requirement to obtain a second biopsyspecimen.

To minimize the possibility of subsequent immunogenic reactions in theengraftment patient, the removal of the various antigenic constituentproteins contained within the serum may be facilitated by collection ofthe fibroblasts by centrifugation, washing the cells repeatedly inphosphate-buffered saline (PBS), and then either re-suspending orculturing the washed fibroblasts for a period of 2-24 hours inserum-free medium containing requisite growth factors which are wellknown in the field. Culture media include, but are not limited to,Fibroblast Basal Medium (FBM). Alternately, the fibroblasts may becultured utilizing the patient's own serum in the appropriate growthmedium.

After the culture has reached a state of confluence, the fibroblasts mayeither be processed for injection or further cultured to facilitate theformation of a three-dimensional “tissue” for subsequent surgicalengraftment. Fibroblasts utilized for injection consist of cellssuspended in a collagen gel matrix. The collagen gel matrix ispreferably comprised of a mixture of 2 ml of a collagen solutioncontaining 0.5 to 1.5 mg/ml collagen in 0.05% acetic acid, 1 ml of DMEMmedium, 270 μl of 7.5% sodium bicarbonate, 48 μl of 100 μg/ml solutionof gentamycin sulfate, and up to 5×10⁶ fibroblast cells/ml of collagengel. Following the suspension of the fibroblasts in the collagen gelmatrix, the suspension is allowed to solidify for approximately 15minutes at room temperature or 37° C. in a 5% Co2 atmosphere. Thecollagen may be derived from human or bovine sources, or from thepatient and may be enzymatically- or chemically-modified (e.g.,atelocollagen).

Three-dimensional “tissue” is formed by initially suspending thefibroblasts in the collagen gel matrix as described above. Preferably,in the culture of three-dimensional tissue, full-length collagen isutilized, rather than truncated or modified collagen derivatives. Theresulting suspension is then placed into a proprietary “transwell”culture system which is typically comprised of culture well in which thelower growth medium is separated from the upper region of the culturewell by a microporous membrane. The microporous membrane typicallypossesses a pore size-ranging from 0.4 to 8 μm in diameter and isconstructed from materials including, but not limited to, polyester,nylon, nitrocellulose, cellulose acetate, polyacrylamide, cross-linkeddextrose, agarose, or other similar materials. The culture wellcomponent of the transwell culture system may be fabricated in anydesired shape or size (e.g., square, round, ellipsoidal, etc.) tofacilitate subsequent surgical tissue engraftment and typically holds avolume of culture medium ranging from 200 μl to 5 ml. In general, aconcentration ranging from 0.5×10⁶ to 10×10⁶ cells/ml, and preferably5×10⁶ cells/ml, are inoculated into the collagen/fibroblast-containingsuspension as described above. Utilizing a preferred concentration ofcells (i.e., 5×10⁶ cells/ml), a total of approximately 4-5 weeks isrequired for the formation of a three-dimensional tissue matrix.However, this time may vary with increasing or decreasing concentrationsof inoculated cells. Accordingly, the higher the concentration of cellsutilized the less time due to a higher overall rate of cellproliferation and replacement of the exogenous collagen with endogenouscollagen and other constituent materials which form the extracellularmatrix synthesized by the cultured fibroblasts. Constituent materialswhich form the extracellular matrix include, but are not limited to,collagen, elastin, fibrin, fibrinogen, proteases, fibronectin, laminin,fibrellins, and other similar proteins. It should be noted that thepotential for immunogenic reaction in the engrafted patient is markedlyreduced due to the fact that the exogenous collagen used in establishingthe initial collagen/fibroblast-containing suspension is graduallyreplaced during subsequent culture by endogenous collagen andextracellular matrix materials synthesized by the fibroblasts.

B. In Vitro Culture of Adipocytes

Adipocytes require a “feeder-layer” or other type of solid support onwhich to grow. One potential solid support may be provided byutilization of the previously discussed collagen gel matrix.Alternately, the solid support may be provided by cultured extracellularmatrix. In general, the in vitro culture of adipocytes is performed bythe mechanical or enzymatic disaggregation of the adipocytes fromadipose tissue derived from a biopsy specimen. The adipocytes are“seeded” onto the surface of the aforementioned solid support andallowed to grow until near-confluence is reached. The adipocytes areremoved by gentle scraping of the solid surface. The isolated adipocytesare then cultured in the same manner as utilized for fibroblasts aspreviously discussed in Section III A.

C. Isolation of the Extracellular Matrix

The extracellular matrix (ECM) may be isolated in either a cellular oracellular form. Constituent materials which form the ECM include, butare not limited to, collagen, elastin, fibrin, fibrinogen, proteases,fibronectin, laminin, fibrellins, and other similar proteins. ECM istypically isolated by the initial culture of cells derived from skin orvocal cord biopsy specimens as previously described. After the culturedcells have reached a minimum of 25-50% sub-confluence, the ECM may beobtained by mechanical, enzymatic, chemical, or denaturant treatment.Mechanical collection is performed by scraping the ECM off of theplastic culture vessel and re-suspending in phosphate-buffered saline(PBS). If desired, the constituent cells are lysed or ruptured byincubation in hypotonic saline containing 5 mM EDTA. Preferably,however, scraping followed by PBS re-suspension is generally utilized.Enzymatic treatment involves brief incubation with a proteolytic enzymesuch as trypsin. Additionally, the use of detergents such as sodiumdodesyl sulfate (SDS) or treatment with denaturants such as urea ordithiotheritol (DTT) followed by dialysis against PBS, will alsofacilitate the release of the ECM from surrounding associated tissue.

The isolated ECM may then be utilized as a “filler” material in thevarious augmentation or repair procedures disclosed in the presentapplication. In addition, the ECM may possess certain cell growth- ormetabolism-promoting characteristics.

D. In Vitro Culture of Fetal or Juvenile Cells or Tissues

In another preferred embodiment, rather than utilizing the patient's owntissue, all of the aforementioned cells, cell suspensions, or tissuesmay be derived from fetal or juvenile sources. Fetal cells lack theimmunogenic determinants responsible for eliciting the hostgraft-rejection reaction and thus may be utilized for engraftmentprocedures with little or no probability of a subsequent immunogenicreaction. An acellular ECM may also be obtained from fetal ECM byhypotonic lysing of the constituent cells. The acellular ECM derivedfrom fetal or juvenile sources or from in vitro culture of early passagecells typically possesses differs in both quantity and characteristicsfrom that of the ECM derived from senescent or late-passage cells. Thecellular or acellular ECM derived from fetal or juvenile sources may beused as a “filler” material in the various augmentation or repairprocedures disclosed in the present application. In addition, the fetalor juvenile ECM may possess certain cell growth- or metabolism-promotingcharacteristics.

E. Injection of Autologous Cultured Dermal/Fascial Fibroblasts

To augment or repair dermal detects, autologously cultured fibroblastsare injected initially into the lower dermis, next in the upper andmiddle dermis, and finally in the subcutaneous regions of the skin as toform raised areas or “wheals.”. The fibroblast suspension is injectedvia a syringe with a needle ranging frog 30 to 18 gauge, with the gaugeof the needle being dependent upon such factors as the overall viscosityof the fibroblast suspension and the type of anesthetic utilized.Preferably, needles ranging from 22 to 18 gauge and 30 to 27 gauge areused with general and local anesthesia, respectively.

To inject the fibroblast suspension into the lower dermis, the needle isplaced at approximately a 45° angle to the skin with the bevel of theneedle directed downward. To place the fibroblast suspension into themiddle dermis the needle is placed at approximately a 20-30° angle. Toplace the suspension into the upper dermis, the needle is placed almosthorizontally (i.e., ˜10-15° angle). Subcutaneous injection isaccomplished by initial placement of the needle into the subcutaneoustissue and injection of the fibroblast suspension during subsequentneedle withdrawal. In addition, it should be noted that the needle ispreferably inserted into the skin from various directions such that theneedle tract will be somewhat different with each subsequent injection.This technique facilitates a greater amount of total skin area receivingthe injected fibroblast suspension.

Following the aforementioned injections, the skin should be expanded andpossess a relatively taut feel. Care should be taken so as not toproduce an overly hard feel to the injected region. Preferably,depressions or rhytids appear elevated following injection and should be“overcorrected” by a slight degree of over-injection of the fibroblastsuspension, as typically some degree of settling or shrinkage will occurpost-operatively.

In some scenarios, the injections may pass into deeper tissue layers.For example, in the case of lip augmentation or repair, a preferredmanner of injection is accomplished by initially injecting thefibroblast suspension into the dermal and subcutaneous layers aspreviously described, into the skin above the lips at the vermillionborder. In addition, the vertical philtrum may also be injected. Thesuspension is subsequently injected into the deeper tissues of the lip,including the muscle, in the manner described for subcutaneousinjection.

F. Surgical Placement of Autologously Cultured Dermal/Fascial FibroblastStrands

In a preferred methodology utilized to augment or repair the skin and/orlips by the surgical placement of autologously cultured dermal and/orfascial fibroblast strands, a needle (the “Passer needle”) is selectedwhich is larger in diameter and greater in length than the area to berepaired or augmented The passer needle is then placed into the skin andthreaded down the length of the area. Guide sutures are placed at bothends' through the dermal or fascial fibroblast strand. One end of theguide suture is fixed to a Keith needle which is subsequently placedthrough the passer needle. The guide suture is brought out through theskin on the side furthest (distal point) from the initial entry point ofthe passer needle. The dermal or fascial fibroblast graft is then pulledinto the passer needle and its position may be adjusted by pulling onthe distal point guide suture or, alternately, the guide suture closestto the passer needle entry point. While the dermal or fascial strand isheld in place by the distal point suture, the passer needle is pulledbackward and removed, thus resulting in the final placement of the graftfollowing the final cutting of the remaining suture.

Generally, the fascial or dermal graft is placed into the subcutaneouslayer of the skin. However, in some situations, it may be placed eithermore deeply or superficially.

If the area to be repaired or augmented is either smaller or larger thanwould be practical to fill with the aforementioned needle method, asubcutaneous “pocket” may be created with a myringotomy knife, scissors,or other similar instrument. A piece of dermis or fascia is thenthreaded into this area by use of guide sutures and passer needle, asdescribed above.

G. Injection of Cells or Other Substances into the Vocal Cords or Larynx

Generally, it is not possible to inject cellular matter or othersubstances directly into the vocal cord epithelium due to its extremethinness. Accordingly, injections are usually made into the laminapropria layer or the muscle itself.

Generally, lamina propria tissue (finely minced if required forinjection), fibroblasts derived from lamina propria tissue, orgelatinous substances are utilized for injection. The preferablemethodology consists of injection directly into the space containing thelamina propria, specifically into Reinkel's space. Injection isaccomplished by use of laryngeal injection needles of the smallestpossible gauge which will accommodate the injectate without the use ofextraneous pressure during the actual injection process. This is asubjective process as to the overall “feel” and the use of too muchpressure may irreparably damage the injected cells. The material isinjected via a syringe with a needle ranging from 30 to 18 gauge, withthe gauge of the needle being dependent upon such factors as the overallviscosity of the injectate and the type of anesthetic utilized.Preferably, needles ranging from 22 to 18 gauge and 30 to 27 gauge areused with general and local anesthesia, respectively. If required,several injections may be performed along the length of the vocal cord.

To medialize a vocal cord with autologously cultured fascial or dermalfibroblasts, the materials are preferably injected directly into thetissue lateral or at the lateral edge of the vocal cord. The fibroblastsmay be injected into scar, Reinke's space, or muscle, depending upon thespecific vocal cord pathology. Preferably, it would be injected into themuscle.

The procedure may be performed under general, local, topical, monitored,or with no anesthesia, depending upon patient compliance and tolerance,the amount of injected material, and the type of injection-performed.

If a greater degree of augmentation is required, a “pocket” may becreated by needle dissection. Alternately, laryngeal microdisection,using knives and dissectors, may be performed. The desired material isthen placed into the pocket with laryngeal forceps, or directlyinjected, depending upon the size of the pocket, the size of the graftmaterial, the anesthesia, and the open access. If the pocket is leftopen after the procedure, it is preferably closed with sutures,adhesive, or a laser, depending upon the size and availability of thesematerials and the individual preferences of the surgeon.

While embodiments and applications of the present invention have beendescribed in some detail by way of illustration and example for purposesof clarity and understanding, it would be apparent to those individualswhom are skilled within the relevant art that many additionalmodifications would be possible without departing from the inventiveconcepts contained herein.

1. A method for corrective surgery in a human subject of a vocal corddefect amenable to rectification by the augmentation of tissuesubadjacent to the vocal cord defect, the method comprising: placing aneffective amount of autologous in vitro cultured cells into a vocal cordtissue of the subject in a position subadjacent to the vocal corddefect, wherein the vocal cord tissue is selected from the groupconsisting of a scar, Reinke's space, a muscle of the vocal cord, andthe lamina propria, wherein the in vitro cultured cells are obtained byculturing a plurality of viable cells retrieved from the subject, andwherein the cultured cells are suspended in a collagen or modifiedcollagen solution prior to injection of the cultured cells.
 2. Themethod of claim 1 wherein the cells are cultured in a collagen ormodified collagen matrix.
 3. The method of claim 2 wherein the cells andthe matrix are placed in the tissue by injection.
 4. The method of claim2 wherein the cells and the matrix are placed in the tissue byengraftment.
 5. The method of claim 1 wherein the in vitro culturedcells are fibroblasts.
 6. The method of claim 5 wherein the fibroblastsare derived from a tissue selected from the group consisting of dermis,fascia, connective tissue, and lamina propria.
 7. The method of claim 1wherein serum from the subject is used for the in vitro culture of thecells.